The culturing of cells for cell banking, for production of cell products, such as recombinant protein production is hampered by changing conditions as cells grow. While stainless steel bioreactors are often used for cell production, disposables are increasingly used at all stages in biologics manufacturing (Rao et al., 2009). In upstream processing, disposable bioreactors offer many advantages over their stainless steel counterparts (ranging from reducing cross-contamination risks to cost and time savings). The WAVE BIOREACTOR™ is a well-documented example of disposable upstream technology used for recombinant protein production in the biopharmaceutical industry (Cronin et al., 2007; Haldankar et al., 2006; Ling et al., 2003; Ye et al., 2009).
The WAVE BIOREACTOR™ system, as developed by Singh (Singh, 1999), comprises a pre-sterilized, flexible and disposable culture chamber (CELLBAG™) CO2- and/or O2-air mix controllers, and a pneumatically-controlled platform for rocking and heating the CELLBAG™. The rocking motion generated by this platform provides mixing and gas transfer in the CELLBAG™.
The WAVE BIOREACTOR™ system can be further equipped to provide online pH and dissolved oxygen (DO) monitoring and real-time feedback control (Mikola et al., 2007; Tang et al., 2007). However, the additional devices required, as well as the need for specially-designed bags to accommodate the pH and DO probes, increase the operational cost and complexity of this system. In addition, the base addition required to raise culture pH to the defined setpoint in pH-controlled bioreactors increases the culture osmolality. Depending on the extent of the osmolality increase in the bioreactor, the associated decrease in cell growth and viability (deZengotita et al., 2002; Zhu et al., 2005) may offset the benefits of pH control. In addition, if the pH probe malfunctions, the resulting pH perturbations may alter cell metabolism and promote cell death (Miller et al., 1988; Osman et al., 2002).
Tight pH and DO controls may not be necessary for certain cell culture applications, such as, for example, the routine passage of cells in small-scale culture systems, such as shake flasks and spinners, for cell maintenance and expansion. However, pH and DO extremes are detrimental to cell growth and viability (Lin et al., 1993; Link et al., 2004; Miller et al., 1988; Osman et al., 2001), and may affect product quality (Restelli et al., 2006; Yoon et al., 2005). Therefore, it is critically important to maintain some control over such growth conditions of cells for all stages of biologics manufacturing. Researchers previously demonstrated success in culturing CHO cells in a pH range of 6.8-7.3 and in the DO range of 10-100% of air saturation (Link et al. 2004; Restelli et al. 2006; Trummer et al. 2006; Yoon et al. 2005).
The added features of conventional bioreactors such as real-time pH monitors and DO monitoring control add significantly to the cost and labor-intensity of cell culture in biological manufacturing. Further, the failure or malfunction of these features can cause unacceptable variations and potential loss of the cell culture which is very costly in time and resources.
Thus, there is a need for improved methods for culturing eukaryotic cells without the need for introduction of strong bases, and without additional monitoring and real-time control of pH and DO.